The primay objective of this project is to determine the mechanism by which TSH regulates thyroid gland function. TSH rapidly stimulates the adenylate cyclase-cyclic AMP system and most, if not all, of the metabolic effects of TSH can be attributed to this cyclic nucleotide. Specific binding of 125I-TSH has been demonstrated using a thyroid plasma membrane fraction which contains a TSH-responsive adenylate cyclase activity. Studies will be done to further characterize such binding and the TSH-receptors. Protein kinase activity is augmented by cyclic AMP in thyroid tissue. The distribution of this enzyme in thyroid subcellular fractions will be determined. Since TSH induces morphologic changes in the apical area of thyroid cell, attempts would be made to subfractionate thyroid plasma membranes into basal and apical membranes by discontinuous sucrose gradient centrifugation. The subfractions would be characterized in relation to TSH binding, adenylate cyclase activity, protein kinase activity and possible substrates, and by electron microscopy. Solubilization of TSH receptors and protein kinase from the membrane would be done. Previous exposure of thyroid tissue to TSH induces refractoriness to the subsequent addition of the hormone when the adenylate cyclase-cyclic AMP system is evaluated. The relationship of this refractoriness to activation of protein kinase, glucose oxidation, iodine metabolism and phospholipid synthesis will be investigated. Since prostaglandins, cholera enterotoxin, dibutyryl cyclic AMP and acetylcholine reproduce some of these effects of TSH, their interaction with TSH to induce refractoriness would be examined.